Methods and reagents for tumor targeting with greater efficacy and less toxicity

ABSTRACT

The present invention relates to a method for treating cancer. This method involves providing a first agent comprising a first targeting component coupled to a first cancer therapeutic component and providing a second agent comprising a second targeting component coupled to a second cancer therapeutic component. The first and second targeting components have different biodistributions and/or pharmacokinetics. The first and second agents are administered to a subject having cancer to treat the cancer. Also disclosed is a combination therapeutic comprising the first and second agents.

This application is a continuation of U.S. patent application Ser. No.16/610,816, filed Nov. 4, 2019, which is a national stage applicationunder 35 U.S.C. § 371 of International Patent Application No.PCT/US2018/030620, filed May 2, 2018, which claims the benefit of U.S.Provisional Patent Application Ser. No. 62/500,187, filed May 2, 2017,which is hereby incorporated by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to methods and reagents for tumortargeting with greater therapeutic efficacy and less toxicity.

BACKGROUND OF THE INVENTION

Combination therapy is a common, accepted treatment approach forvirtually all types of cancers and has been the standard therapeuticapproach for several decades. The basis for the adoption of combinationtherapy was the early chemotherapy experience where it was determinedthat the high mutational rate of cancers allowed rapid development ofresistant strains of tumor cells when only a single agent was employed.The goal of combination therapies is to increase efficacy and minimizethe development of tumor resistance or escape. This is generallyachieved by employing 2 or more anti-cancer agents each of which has adifferent mechanism of action, making the development of resistant tumorcells more difficult and less likely. The additive or synergisticeffects of combining two or more agents can be the difference betweensuccessful and unsuccessful treatment of the patient.

Many combination treatment regimens are well known in the oncologyfield. As an example, MOPP (an acronym for mechlorethamine, vincristine,procarbazine, prednisone) is a curative treatment regimen for Hodgkins'Disease. Several different combination regimens (which all includecisplatin, vinblastine, and bleomycin) are accepted in the treatment oftesticular cancer, which is curable in up to 98% of diagnosed cases. Inall, more than 300 different combination regimens have been used.

The main drawback to combination therapy is often that it also resultsin an increase in toxicity. For example, most forms of nonsurgicalcancer therapy, such as external irradiation and chemotherapy, arelimited in their efficacy because of toxic side effects to normaltissues and cells as well as the limited specificity of these treatmentmodalities for cancer cells. This limitation is also of importance whenanti-cancer antibodies are used for targeting toxic agents, such asisotopes, drugs, and toxins, to cancer sites, because, as systemicagents, they also circulate to sensitive cellular compartments such asthe bone marrow. In acute radiation injury, there is destruction oflymphoid and hematopoietic compartments as a major factor in thedevelopment of septicemia and subsequent death. Thus, methods ofreducing the toxic effects of cancer therapy while maintaining or evenincreasing efficacy are in high demand.

The present invention is directed to overcoming these and otherdeficiencies in the art.

SUMMARY OF THE INVENTION

The present invention relates to a method of treating cancer. Thismethod involves providing a first agent comprising a first targetingcomponent coupled to a first cancer therapeutic component and providinga second agent comprising a second targeting component coupled to asecond cancer therapeutic component. The first and second targetingcomponents have different biodistributions and/or pharmacokinetics. Thefirst and second agents are then administered, to a subject havingcancer, to treat the cancer.

The present invention also pertains to a combination therapeutic fortreating cancer. The combination therapeutic includes a first agentcomprising a first targeting component coupled to a first cancertherapeutic component and a second agent comprising a second targetingcomponent coupled to a second cancer therapeutic component. The firstand second targeting components have different biodistributions and/orpharmacokinetics.

The present invention has devised a way to overcome the MTD of atargeted agent in order to achieve improved efficacy with no increasein, and an opportunity to decrease, its toxicity. The present inventionproposes the use of two individual targeting agents, rather than one,each targeting the same molecule or cell type. In this approach, each ofthe two targeted agents has a different biodistribution and/orpharmacokinetics from the other. Importantly, the differentbiodistributions and pharmacokinetics of these respective agents resultsin differing, non-overlapping toxicities of each of the two respectivetargeted agents. When the two targeted agents are combined in atreatment strategy, the result is that both drugs converge,simultaneously or sequentially, at the desired target site therebyproviding a combined treatment effect. The normal tissue toxicity,however, is not increased as the biodistribution of the two targetedagents differs such that there is no increase in drug delivery to thenormal tissues and, therefore, no increase in toxicity.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1B show that co-incubation of radiolabeled anti-PSMA antibodyJ591-¹⁷⁷Lu and radiolabeled PSMA-617-¹⁷⁷Lu (a small molecule PSMAinhibitor) results in additive ¹⁷⁷Lu internalization in PSMA-positivecells in vitro. FIG. 1A shows ¹⁷⁷Lu internalization in LNCaP cells. FIG.1B shows ¹⁷⁷Lu internalization in CWR22Rv1 cells.

FIG. 2 shows that an anti-tumor effect could be achieved in LNCaPxenografts at half the dose of J591-¹⁷⁷Lu (i.e., 75 μCi) by also addinga dose of PSMA-617-¹⁷⁷Lu (i.e., 200 μCi) well below its MTD.

FIG. 3 shows the anti-tumor response of J591-¹⁷⁷Lu at its MTD could beequaled by J591-¹⁷⁷Lu at 135 μCi (75% of MTD) plus 525 μCi (a sub-MTDdose) of PSMA-617-¹⁷⁷Lu.

FIG. 4 shows expression of PSMA in human salivary gland alveoli.

FIGS. 5A-5B show expression of PSMA in human salivary gland intercalatedducts at 20× magnification (FIG. 5A) and at 10× magnification (FIG. 5B).

FIGS. 6A-6B show expression of PSMA in human kidney at 4× magnification(FIG. 6A) and at 10× magnification (FIG. 6B).

FIG. 7 shows expression of PSMA in small bowel.

FIGS. 8A-8B show the bio-distribution of PSMA peptides/inhibitors bypositron emission tomography with 68 Ga-PSMA 11 (FIG. 8A)(Afshar-Oromieh et al., “PET Imaging With a [68Ga]Gallium-Labelled PSMALigand for the Diagnosis of Prostate Cancer: Biodistribution in Humansand First Evaluation of Tumour Lesions,” Eur. J. Nucl. Med. Mol. Imaging40(4):486-95 (2013), which is hereby incorporated by reference in itsentirety) and 68 Ga-PSMA I&T (FIG. 8B) (Herrmann et. al.,“Biodistribution and Radiation Dosimetry for a Probe TargetingProstate-Specific Membrane Antigen for Imaging and Therapy,” J. Nucl.Med. 56(6):855-61 (2015), which is hereby incorporated by reference inits entirety).

FIGS. 9A-9B show the bio-distribution of Anti-PSMA antibodies byanterior (FIG. 9A) and posterior (FIG. 9B) positron emission tomography.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a method of treating cancer. Thismethod involves providing a first agent comprising a first targetingcomponent coupled to a first cancer therapeutic component and providinga second agent comprising a second targeting component coupled to asecond cancer therapeutic component. The first and second targetingcomponents have different biodistributions and/or pharmacokinetics. Thefirst and second agents are then administered, to a subject havingcancer, to treat the cancer.

As used herein, the term “treat” refers to the application oradministration of the first and second agents of the invention to asubject, e.g., a patient. The treatment can be to cure, heal, alleviate,relieve, alter, remedy, ameliorate, palliate, improve or affect thecancer, the symptoms of the cancer or the predisposition toward thecancer.

As used herein, the term “subject” is intended to include human andnon-human animals. Non-human animals include all vertebrates, e.g.,mammals and non-mammals, such as non-human primates, sheep, dog, cow,chickens, amphibians, reptiles, etc.

As used herein, the term “cancer” includes all types of cancerousgrowths or oncogenic processes, metastatic tissues or malignantlytransformed cells, tissues, or organs, irrespective of histopathologictype or stage of invasiveness.

As used herein, the terms “maximum tolerated dose (MTD)” refers to thedose of any therapeutic drug—including targeted drugs—above whichunacceptable toxicity occurs. This is true whether the drugs aretargeted to a particular cell type or a particular molecule. Because ofthe MTD and the limit of tolerability of a drug (targeted or otherwise),maximal anti-cancer efficacy is generally not attainable. The MTD of adrug is impacted significantly by its biodistribution and itspharmacokinetics.

As used herein, the term “biodistribution” refers to the organs andtissues to which a drug distributes in the body.

As used herein, the term “pharmacokinetics” refers to how long a drugstays in the body.

In certain embodiments, the cancer is prostate cancer, neuroendocrinecancer, breast cancer, or non-Hodgkin's lymphoma. In some embodiments,the cancer is a primary tumor, while in other embodiments, the cancer isa secondary or metastatic tumor.

As used herein, the “targeting component” is a component that is able tobind to or otherwise associate with a molecular target, for example, amembrane component, a cell surface receptor, prostate specific membraneantigen (PSMA, which is also known as folate hydrolase 1, glutamatecarboxypeptidase II, and NAALADase), or the like. A first and secondagent comprising the targeting component may become localized orconverge at a particular targeted site, for instance, a tumor, a diseasesite, a tissue, an organ, a type of cell, etc. As such, the first andsecond agent may be “target-specific.” In some cases, the therapeuticagent may exert its anti-cancer effect without the need for release fromthe targeting component. In other cases, the therapeutic component maybe released from the first and/or second agent and allowed to interactlocally with the particular targeting site.

For example, contemplated targeting components may include a nucleicacid, peptide, polypeptide, protein, glycoprotein, carbohydrate, orlipid. A targeting component may be a naturally occurring or syntheticligand for a cell surface receptor, e.g., a growth factor, hormone, LDL,transferrin, etc. A targeting component can be an antibody, which termis intended to include antibody fragments, characteristic portions ofantibodies, single chain targeting moieties which can be identified, forexample, using procedures such as phage display. Targeting componentsmay also be a targeting peptide, targeting peptidomimetic, or a smallmolecule, whether naturally-occurring or artificially created (e.g., viachemical synthesis).

In one embodiment, the first and second targeting components areindependently selected from the group consisting of an antibody orbinding fragment thereof, a protein, a peptide, oligonucleotide, and asmall molecule.

Antibodies against molecular targets on tumors are known. For example,antibodies and antibody fragments which specifically bind markersproduced by or associated with tumors have been disclosed, inter alia,in U.S. Pat. No. 3,927,193 to Hansen, and U.S. Pat. Nos. 4,331,647,4,348,376, 4,361,544, 4,468,457, 4,444,744, 4,818,709 and 4,624,846 toGoldenberg, the contents of all of which are incorporated herein byreference in their entirety. In particular, antibodies against anantigen, e.g., a gastrointestinal, lung, breast, prostate, ovarian,testicular, brain or lymphatic tumor, a sarcoma or a melanoma, areadvantageously used. Antibodies to cancer-related antigens are wellknown to those in the art.

The antibodies of the present invention may exist in a variety of formsincluding, for example, polyclonal antibodies, monoclonal antibodies,intracellular antibodies (“intrabodies”), antibody fragments (e.g. Fv,Fab and F(ab)2), half-antibodies, hybrid derivatives, as well as singlechain antibodies (scFv), chimeric antibodies and humanized antibodies(Ed Harlow and David Lane, USING ANTIBODIES: A LABORATORY MANUAL (ColdSpring Harbor Laboratory Press, 1999); Houston et al., “ProteinEngineering of Antibody Binding Sites: Recovery of Specific Activity inan Anti-Digoxin Single-Chain Fv Analogue Produced in Escherichia coli,”Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988); Bird et al,“Single-Chain Antigen-Binding Proteins,” Science 242:423-426 (1988),each of which is hereby incorporated by reference in its entirety).

Antibodies of the present invention may also be synthetic antibodies. Asynthetic antibody is an antibody which is generated using recombinantDNA technology, such as, for example, an antibody expressed by abacteriophage. Alternatively, the synthetic antibody is generated by thesynthesis of a DNA molecule encoding and expressing the antibody of thepresent invention or the synthesis of an amino acid sequence specifyingthe antibody, where the DNA or amino acid sequence has been obtainedusing synthetic DNA or amino acid sequence technology which is availableand well known in the art.

Methods for monoclonal antibody production may be carried out using thetechniques described herein or are well-known in the art (MONOCLONALANTIBODIES—PRODUCTION, ENGINEERING AND CLINICAL APPLICATIONS (Mary A.Ritter and Heather M. Ladyman eds., 1995), which is hereby incorporatedby reference in its entirety). Generally, the process involves obtainingimmune cells (lymphocytes) from the spleen of a mammal which has beenpreviously immunized with the antigen of interest either in vivo or invitro.

Alternatively monoclonal antibodies can be made using recombinant DNAmethods as described in U.S. Pat. No. 4,816,567 to Cabilly et al, whichis hereby incorporated by reference in its entirety. The polynucleotidesencoding a monoclonal antibody are isolated from mature B-cells orhybridoma cells, for example, by RT-PCR using oligonucleotide primersthat specifically amplify the genes encoding the heavy and light chainsof the antibody. The isolated polynucleotides encoding the heavy andlight chains are then cloned into suitable expression vectors, whichwhen transfected into host cells such as E. coli cells, simian COScells, Chinese hamster ovary (CHO) cells, or myeloma cells that do nototherwise produce immunoglobulin protein, monoclonal antibodies aregenerated by the host cells. Also, recombinant monoclonal antibodies orfragments thereof of the desired species can be isolated from phagedisplay libraries (McCafferty et al., “Phage Antibodies: FilamentousPhage Displaying Antibody Variable Domains,” Nature 348:552-554 (1990);Clackson et al., “Making Antibody Fragments using Phage DisplayLibraries,” Nature 352:624-628 (1991); and Marks et al., “By-PassingImmunization. Human Antibodies from V-Gene Libraries Displayed onPhage,” J. Mol. Biol. 222:581-597 (1991), which are hereby incorporatedby reference in their entirety).

The polynucleotide(s) encoding a monoclonal antibody can further bemodified using recombinant DNA technology to generate alternativeantibodies. For example, the constant domains of the light and heavychains of a mouse monoclonal antibody can be substituted for thoseregions of a human antibody to generate a chimeric antibody.Alternatively, the constant domains of the light and heavy chains of amouse monoclonal antibody can be substituted for a non-immunoglobulinpolypeptide to generate a fusion antibody. In other embodiments, theconstant regions are truncated or removed to generate the desiredantibody fragment of a monoclonal antibody. Furthermore, site-directedor high-density mutagenesis of the variable region can be used tooptimize specificity and affinity of a monoclonal antibody.

The monoclonal antibody of the present invention can be a humanizedantibody. Humanized antibodies are antibodies that contain minimalsequences from non-human (e.g., murine) antibodies within the variableregions. Such antibodies are used therapeutically to reduce antigenicityand human anti-mouse antibody responses when administered to a humansubject. In practice, humanized antibodies are typically humanantibodies with minimal to no non-human sequences. A human antibody isan antibody produced by a human or an antibody having an amino acidsequence corresponding to an antibody produced by a human.

In addition to whole antibodies, the present invention encompassesbinding portions of such antibodies. Such binding portions include themonovalent Fab fragments, Fv fragments (e.g., single-chain antibody,scFv), and single variable V_(H) and V_(L) domains, and the bivalentF(ab′)2 fragments, Bis-scFv, diabodies, triabodies, minibodies, etc.These antibody fragments can be made by conventional procedures, such asproteolytic fragmentation procedures, as described in James Goding,MONOCLONAL ANTIBODIES: PRINCIPLES AND PRACTICE 98-118 (Academic Press,1983) and Ed Harlow and David Lane, ANTIBODIES: A LABORATORY MANUAL(Cold Spring Harbor Laboratory, 1988), which are hereby incorporated byreference in their entirety, or other methods known in the art.

It may further be desirable, especially in the case of antibodyfragments, to modify the antibody in order to increase its serumhalf-life. This can be achieved, for example, by incorporation of asalvage receptor binding epitope into the antibody fragment by mutationof the appropriate region in the antibody fragment or by incorporatingthe epitope into a peptide tag that is then fused to the antibodyfragment at either end or in the middle (e.g., by DNA or peptidesynthesis).

Antibody mimics are also suitable for use in accordance with the presentinvention. A number of antibody mimics are known in the art including,without limitation, those known as monobodies, which are derived fromthe tenth human fibronectin type III domain (¹⁰Fn3) (Koide et al., “TheFibronectin Type III Domain as a Scaffold for Novel Binding Proteins,”J. Mol. Biol. 284:1141-1151 (1998); Koide et al., “Probing ProteinConformational Changes in Living Cells by Using Designer BindingProteins: Application to the Estrogen Receptor,” Proc. Natl. Acad. Sci.USA 99:1253-1258 (2002), each of which is hereby incorporated byreference in its entirety); and those known as affibodies, which arederived from the stable alpha-helical bacterial receptor domain Z ofstaphylococcal protein A (Nord et al., “Binding Proteins Selected fromCombinatorial Libraries of an alpha-helical Bacterial Receptor Domain,”Nature Biotechnol. 15(8):772-777 (1997), which is hereby incorporated byreference in its entirety).

The peptides used in conjunction with the present invention can beobtained by known isolation and purification protocols from naturalsources, can be synthesized by standard solid or solution phase peptidesynthesis methods according to the known peptide sequence of thepeptide, or can be obtained from commercially available preparations.Included herein are peptides that exhibit the biological bindingproperties of the native peptide and retain the specific bindingcharacteristics of the native peptide. Derivatives and analogs of thepeptide, as used herein, include modifications in the composition,identity, and derivitization of the individual amino acids of thepeptide provided that the peptide retains the specific bindingproperties of the native peptide. Examples of such modifications wouldinclude modification of any of the amino acids to include theD-stereoisomer, substitution in the aromatic side chain of an aromaticamino acid, derivitization of the amino or carboxyl groups in the sidechains of an amino acid containing such a group in a side chain,substitutions in the amino or carboxy terminus of the peptide, linkageof the peptide to a second peptide or biologically active moiety, andcyclization of the peptide (G. Van Binst and D. Tourwe, “BackboneModifications in Somatostatin Analogues: Relation Between Conformationand Activity,” Peptide Research 5:8-13 (1992), which is herebyincorporated by reference in its entirety).

In one embodiment, the first and second targeting components target thesame molecular target. For example, the first and second targetingcomponents may bind to the same receptor (e.g. PSMA) expressed by thesame cell type.

In another embodiment, the first and second targeting components targetdifferent molecular targets on the same cell type. For example, thefirst and second targeting components may bind to different receptors(e.g. HER1 and HER2) expressed on the same cell type.

As used herein, the “cancer therapeutic component” is an agent, orcombination of agents, that treats a cell, tissue, or subject having acondition requiring therapy, when contacted with the cell, tissue orsubject. The first and second cancer therapeutic components may be thesame or different, and may be, for example, therapeutic radionuclides,chemotherapeutic agents, hormones, hormone antagonists, receptorantagonists, enzymes or proenzymes activated by another agent,biologics, autocrines or cytokines. Toxins also can be used in themethods of the present invention. Other therapeutic agents useful in thepresent invention include anti-DNA, anti-RNA, radiolabeledoligonucleotides, such as anti-sense oligodeoxy ribonucleotides,anti-protein and anti-chromatin cytotoxic or antimicrobial agents. Othertherapeutic agents are known to those skilled in the art, and the use ofsuch other therapeutic agents in accordance with the present inventionis specifically contemplated.

While the first and second cancer therapeutic components may be thesame, in one embodiment they are different. For example, the first andsecond cancer therapeutic components may comprise differentradionuclides, or the first cancer therapeutic component may comprise achemotherapeutic agent while the second cancer therapeutic componentcomprises a radionuclide, or the first cancer therapeutic component maycomprise a radionuclide while the second cancer therapeutic componentcomprises a chemotherapeutic agent.

In one embodiment, the first and second cancer therapeutic componentsare independently selected from the group consisting of a radionuclideand a chemotherapeutic agent.

In one embodiment, the first and/or second cancer therapeutic componentis a radionuclide independently selected from the group consisting of⁸⁶Re, ⁹⁰Y, ⁶⁷Cu, ¹⁶⁹Er, ¹²¹Sn, ¹²⁷Te, ¹⁴²Pr, ¹⁴³Pr, ¹⁹⁸Au, ¹⁹⁹Au, ¹⁶¹Tb,¹⁰⁹Pd, ¹⁸⁸Rd, ¹⁶⁶Dy, ¹⁶⁶Ho, ¹⁴⁹Pm, ¹⁵¹Pm, ¹⁵³Sm, ¹⁵⁹Gd, ¹⁷²Tm, ¹⁶⁹Yb,¹⁷⁵Yb, ¹⁷⁷Lu, ¹⁰⁵Rh, ¹¹¹Ag, ¹³¹I, ¹⁷⁷mSn, ²²⁵Ac, ²²⁷Th, ²¹¹At, andcombinations thereof.

Procedures for labeling agents with radioactive isotopes are generallyknown in the art. For example, there are a wide range of moieties whichcan serve as chelating ligands and which can be derivatized to thetargeting component of the invention. For instance, the chelating ligandcan be a derivative of 1,4,7,10-tetraazacyclododecanetetraacetic acid(DOTA), ethylenediaminetetraacetic acid (EDTA),diethylenetriaminepentaacetic acid (DTPA), and1-p-Isothiocyanato-benzyl-methyl-diethylenetriaminepentaacetic acid(ITC-MX). These chelators typically have groups on the side chain bywhich the chelator can be used for attachment to a targeting componentof the present invention. Such groups include, e.g.,benzylisothiocyanate, by which the DOTA, DTPA, or EDTA can be coupledto, e.g., an amine group of the targeting component. Procedures foriodinating biological agents, such as antibodies, binding portionsthereof, probes, or ligands, are described by Hunter and Greenwood,“Preparation of Iodine-131 Labelled Human Growth Hormone of HighSpecific Activity,” Nature 144:496-496 (1962), David et al., “ProteinIodination With Solid State Lactoperoxidase,” Biochemistry 13:1014-1021(1974), and U.S. Pat. No. 3,867,517 to Ling and U.S. Pat. No. 4,376,110to David, which are hereby incorporated by reference in their entirety.Other procedures for iodinating biological agents are described byGreenwood et al., “The Preparation of I-131-Labelled Human GrowthHormone of High Specific Radioactivity,” Biochem. J. 89:114-123 (1963);Marchalonis, “An Enzymic Method for the Trace Iodination ofImmunoglobulins and Other Proteins,” Biochem. J. 113:299-305 (1969); andMorrison et al., “Use of Lactoperoxidase Catalyzed Iodination inImmunochemical Studies,” Immunochemistry 8:289-297 (1971), which arehereby incorporated by reference in their entirety. Procedures for^(99m)Tc-labeling are described by Rhodes, B. et al. in Burchiel, S. etal. (eds.), Tumor Imaging: The Radioimmunochemical Detection of Cancer,New York: Masson 111-123 (1982) and the references cited therein, whichare hereby incorporated by reference in their entirety. Proceduressuitable for 111 In-labeling biological agents are described byHnatowich et al., “The Preparation of DTPA-coupled AntibodiesRadiolabeled With Metallic Radionuclides: an Improved Method,” J. Immul.Methods 65:147-157 (1983), Hnatowich et al., “Coupling Antibody WithDTPA—an Alternative to the Cyclic Anhydride,” Int. J. Applied Radiation35:554-557 (1984), and Buckley et al., “An Efficient Method ForLabelling Antibodies With 111In,” F.E.B.S. 166:202-204 (1984), which arehereby incorporated by reference in their entirety.

In another embodiment, the first and/or second cancer therapeuticcomponent is a chemotherapeutic agent independently selected from thegroup consisting of busulfan, cisplatin, carboplatin, chlorambucil,cyclophosphamide, ifosfamide, dacarbazine (DTIC), mechlorethamine(nitrogen mustard), melphalan carmustine (BCNU), lomustine (CCNU),5-fluorouracil (5-FU), capecitabine, methotrexate, gemcitabine,cytarabine (ara-C), fludarabine dactinomycin, daunorubicin, doxorubicin(Adriamycin), idarubicin, mitoxantrone, paclitaxel, docetaxel, etoposide(VP-16), vinblastine, vincristine, vinorelbine prednisone,dexamethasone, tamoxifen, fulvestrant, anastrozole, letrozole, megestrolacetate, bicalutamide, flutamide, leuprolide, goserelin, L-asparaginase,tretinoin, maytansines, auristatins, pyrrolobenzodiazepines,duocarmycins, and combinations thereof.

Procedures for conjugating biological agents with chemotherapeuticagents are well known in the art. Most of the chemotherapeutic agentscurrently in use in treating cancer possess functional groups that areamenable to chemical crosslinking directly with an amine or carboxylgroup of a targeting component of the present invention. For example,free amino groups are available on methotrexate, doxorubicin,daunorubicin, cytosinarabinoside, cisplatin, vindesine, mitomycin, andbleomycin while free carboxylic acid groups are available onmethotrexate, melphalan, and chlorambucil. These functional groups, thatis free amino and carboxylic acids, are targets for a variety ofhomo-bifunctional and hetero-bifunctional chemical crosslinking agentswhich can crosslink these drugs directly to a free amino group of atargeting component. Specific procedures for conjugating targetingcomponents with chemotherapeutic agents have been described and areknown in the art. By way of example, conjugation of chlorambucil withantibodies is described by Flechner, “The Cure and ConcomitantImmunization of Mice Bearing Ehrlich Ascites Tumors by Treatment With anAntibody—Alkylating Agent Complex,” European Journal of Cancer 9:741-745(1973); Ghose et al., “Immunochemotherapy of Cancer withChlorambucil-Carrying Antibody,” British Medical Journal 3:495-499(1972); and Szekerke et al., “The Use of Macromolecules as Carriers ofCytotoxic Groups (part II) Nitrogen Mustard—Protein Complexes,”Neoplasma 19:211-215 (1972), which are hereby incorporated by referencein their entirety. Procedures for conjugating daunomycin and adriamycinto antibodies are described by Hurwitz et al., “The Covalent Binding ofDaunomycin and Adriamycin to Antibodies, With Retention of Both Drug andAntibody Activities,” Cancer Research 35:1175-1181 (1975) and Arnon etal. Cancer Surveys 1:429-449 (1982), which are hereby incorporated byreference in their entirety. Coupling procedures as also described in EP86309516.2, which is hereby incorporated by reference in its entirety.

It will be appreciated that the exact dosage of the first and secondagents of the invention is chosen by the individual physician in view ofthe patient to be treated. In general, dosage and administration areadjusted to provide an effective amount of the agent to the patientbeing treated. As used herein, the “effective amount” of an agent refersto the amount necessary to elicit the desired biological response. Aswill be appreciated by those of ordinary skill in this art, theeffective amount of agent of the invention may vary depending on suchfactors as the desired biological endpoint, the drug to be delivered,the target tissue, the route of administration, etc. For example, theeffective amount of agent containing an anti-cancer drug might be theamount that results in a reduction in tumor size by a desired amountover a desired period of time. Additional factors which may be takeninto account include the severity of the disease state; age, weight andgender of the patient being treated; diet, time and frequency ofadministration; drug combinations; reaction sensitivities; andtolerance/response to therapy.

In general, doses can range from about 25% to about 100% of the MTD ofthe targeted agent when given as a single agent. Based upon thecomposition, the dose can be delivered once, continuously, such as bycontinuous pump, or at periodic intervals. Dosage may be adjustedappropriately to achieve desired drug levels, locally, or systemically.In the event that the response in a subject is insufficient at suchdoses, even higher doses (or effective higher doses by a different, morelocalized delivery route) may be employed to the extent that patienttolerance permits. Continuous IV dosing over, for example, 24 hours ormultiple doses per day also are contemplated to achieve appropriatesystemic levels of compounds.

In one embodiment, the first and second cancer therapeutic componentseach have a maximum tolerated dose, and the maximum tolerated doses ofthe first and second cancer therapeutic components are administered tothe subject. Because the biodistribution and pharmacokinetics aredifferent for the two targeting components, their toxicities asindividual drugs are non- or minimally overlapping. As a result, theincreased, additive dose to the target site is not accompanied by acommensurate increase in toxicity.

In an alternative embodiment, less than the maximum tolerated doses ofthe first and second cancer therapeutic components are administered tothe subject. When the two therapeutic components are combined in atreatment strategy in amounts less than the maximum tolerated dose, theresult is that both drugs converge (simultaneously or sequentially) atthe desired target site thereby providing an additive treatment effectbut, because the agents are administered at less than their MTD, lowertoxicity is experienced by the subject.

In one embodiment, the first agent is an antibody conjugated to aradionuclide and is administered at a dose of about 100 to 160 mCi totalin a 2 week cycle, such as a dose of 100, 110, 120, 130, 140, 150, or160 mCi total in a 2 week cycle.

In another embodiment, the first agent is an antibody conjugated to aradionuclide and is administered at a dose of about 120 to 140 mCi totalin a 2 week cycle, such as a dose of 120, 125, 130, or 140 mCi total ina 2 week cycle.

In a further embodiment, the second agent is a small molecule conjugatedto a radionuclide and is administered at a dose of about 300 to 500 mCitotal in a 2 week cycle, such as a dose of 300, 325, 350, 375, 400, 425,450, 475, or 500 mCi total in a 2 week cycle.

In practicing the methods of the present invention, the administeringstep is carried out to treat cancer in a subject. In one embodiment, asubject having cancer is selected prior to the administering step. Suchadministration can be carried out systemically or via direct or localadministration to the tumor site. By way of example, suitable modes ofsystemic administration include, without limitation orally, topically,transdermally, parenterally, intradermally, intramuscularly,intraperitoneally, intravenously, subcutaneously, or by intranasalinstillation, by intracavitary or intravesical instillation,intraocularly, intraarterialy, intralesionally, or by application tomucous membranes. Suitable modes of local administration include,without limitation, catheterization, implantation, direct injection,dermal/transdermal application, or portal vein administration torelevant tissues, or by any other local administration technique, methodor procedure generally known in the art. The mode of affecting deliveryof agent will vary depending on the type of therapeutic agent (e.g., anantibody or an inhibitory nucleic acid molecule) and the disease to betreated.

The agents of the present invention may be orally administered, forexample, with an inert diluent, or with an assimilable edible carrier,or it may be enclosed in hard or soft shell capsules, or it may becompressed into tablets, or they may be incorporated directly with thefood of the diet. Agents of the present invention may also beadministered in a time release manner incorporated within such devicesas time-release capsules or nanotubes. Such devices afford flexibilityrelative to time and dosage. For oral therapeutic administration, theagents of the present invention may be incorporated with excipients andused in the form of tablets, capsules, elixirs, suspensions, syrups, andthe like. Such compositions and preparations should contain at least0.1% of the agent, although lower concentrations may be effective andindeed optimal. The percentage of the agent in these compositions may,of course, be varied and may conveniently be between about 2% to about60% of the weight of the unit. The amount of an agent of the presentinvention in such therapeutically useful compositions is such that asuitable dosage will be obtained.

When the agents of the present invention are administered parenterally,solutions or suspensions of the agent can be prepared in water suitablymixed with a surfactant such as hydroxypropylcellulose. Dispersions canalso be prepared in glycerol, liquid polyethylene glycols, and mixturesthereof in oils. Illustrative oils are those of petroleum, animal,vegetable, or synthetic origin, for example, peanut oil, soybean oil, ormineral oil. In general, water, saline, aqueous dextrose and relatedsugar solution, and glycols, such as propylene glycol or polyethyleneglycol, are preferred liquid carriers, particularly for injectablesolutions. Under ordinary conditions of storage and use, thesepreparations contain a preservative to prevent the growth ofmicroorganisms.

Pharmaceutical formulations suitable for injectable use include sterileaqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. In all cases, the form must be sterile and must be fluid tothe extent that easy syringability exists. It must be stable under theconditions of manufacture and storage and must be preserved against thecontaminating action of microorganisms, such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquidpolyethylene glycol), suitable mixtures thereof, and vegetable oils.

When it is desirable to deliver the agents of the present inventionsystemically, they may be formulated for parenteral administration byinjection, e.g., by bolus injection or continuous infusion. Formulationsfor injection may be presented in unit dosage form, e.g., in ampoules orin multi-dose containers, with an added preservative. The compositionsmay take such forms as suspensions, solutions or emulsions in oily oraqueous vehicles, and may contain formulatory agents such as suspending,stabilizing and/or dispersing agents.

Intraperitoneal or intrathecal administration of the agents of thepresent invention can also be achieved using infusion pump devices. Suchdevices allow continuous infusion of desired compounds avoiding multipleinjections and multiple manipulations.

In addition to the formulations described previously, the agents mayalso be formulated as a depot preparation. Such long acting formulationsmay be formulated with suitable polymeric or hydrophobic materials (forexample as an emulsion in an acceptable oil) or ion exchange resins, oras sparingly soluble derivatives, for example, as a sparingly solublesalt

According to one embodiment of the present invention, the cancer isprostate cancer.

In another embodiment of this aspect of the invention, when the canceris prostate cancer, the first and second targeting components target thePSMA receptor.

As used herein, “PSMA” or “prostate-specific membrane antigen” proteinrefers to mammalian PSMA, preferably human PSMA protein. The longtranscript of PSMA encodes a protein product of about 100-120 kDamolecular weight characterized as a type II transmembrane receptorhaving sequence homology with the transferrin receptor and havingNAALADase activity (Carter et al., “Prostate-Specific Membrane Antigenis a Hydrolase With Substrate and Pharmacologic Characteristics of aNeuropeptidase,” Proc. Natl. Acad. Sci. USA 93:749-753 (1996), which ishereby incorporated by reference in its entirety).

In an alternative embodiment, the first targeting component is a PSMAreceptor antibody and the second targeting component is a PSMA receptorbinding peptide or PSMA receptor inhibitor.

A PSMA receptor antibody is an antibody that interacts with (e.g., bindsto) PSMA, preferably human PSMA protein. Preferably, the PSMA receptorantibody interacts with, e.g., binds to, the extracellular domain ofPSMA, e.g., the extracellular domain of human PSMA located at aboutamino acids 44-750 of human PSMA (amino acid residues correspond to thehuman PSMA sequence disclosed in U.S. Pat. No. 5,538,866, which ishereby incorporated by reference in its entirety). PSMA receptorantibodies are known in the art (Goldsmith et al., “TargetedRadionuclide Therapy for Prostate Cancer,” in Therapeutic NuclearMedicine 617-628 (R. Baum ed. 2014), which is hereby incorporated byreference in its entirety). Exemplary PSMA receptor antibodies include,but are not limited to, J591, J415, J533, and E99.

The PSMA receptor inhibitor may include any lipids, carbohydrates,polynucleotides, peptides, polypeptides, or any other biologic, organicor inorganic molecules which inhibit the function of the PSMA receptor.Exemplary PSMA receptor inhibitor are known in the art include, but arenot limited to, PSMA 617, PSMA I&T, DCFBC, DCFPyL, glutamate-urea-lysineanalogs, phosphoramidate analogs, and 2-(phosphinylmethyl) pentanedioicacid analogs (Lutje et al., “PSMA Ligands for Radionuclide Imaging andTherapy of Prostate Cancer: Clinical Status,” Theranostics5(12):1388-1401 (2015); Haberkorn et al., “New Strategies in ProstateCancer: Prostate-Specific Membrane Antigen (PSMA) Ligands for Diagnosisand Therapy,” Clin. Cancer Res. 22(1):9-15 (2016), which are herebyincorporated by reference in their entirety).

In one embodiment, the PSMA receptor antibody is selected from the groupconsisting of J591, J415, J533, and E99, while the second targetingcomponent is a peptide selected from the group consisting of PSMA 617,PSMA I&T, DCFBC, DCFPyL, glutamate-urea-lysine analogs, phosphoramidateanalogs, 2-(phosphinylmethyl) pentanedioic acid analogs, and other PSMAligands/inhibitors.

In one embodiment, the first agent is J591-¹⁷⁷Lu and the second agent isPSMA 617-¹⁷⁷Lu or PSMA I&T-¹⁷⁷Lu. By way of example, the PSMA receptorantibodies can be radiolabeled with ¹¹¹Indium, ⁹⁰Yttrium, or ¹⁷⁷Lutetiumby coupling with 1,4,7,10-tetraazacyclododecane-N,N′,N″,N″′-tetraaceticacid (DOTA) as described in U.S. Pat. No. 7,045,605 to Bander, which ishereby incorporated by reference in its entirety.

In another embodiment of the present invention, the cancer is aneuroendocrine cancer. Neuroendocrine cancers include, but are notlimited to, carcinoid tumors, gastrinoma, insulinoma, glucagonoma,VIPoma, somatostatinoma, thyroid carcinoma, Merkel cell carcinoma of theskin, tumor of the anterior pituitary, medullary carcinoma, parathyroidtumor, thymus and mediastinal carcinoid tumor, pulmonary neuroendocrinetumor, adrenomedullary tumor, pheochromocytoma, Schwannoma,paraganglioma, neuroblastoma, and urinary tract carcinoid neuroendocrinecarcinoma.

In accordance with this aspect of the present invention, in oneembodiment, the first and second targeting components target thesomatostatin receptor.

At least five somatostatin receptors subtypes have been characterizedand tumors can express various receptor subtypes (Shaer et al.,“Somatostatin Receptor Subtypes sst1, sst2, sst3 and sst5 Expression inHuman Pituitary, Gastroentero-Pancreatic and Mammary tumors: Comparisonof mRNA Analysis With Receptor Autoradiography,” Int. J. Cancer70:530-537 (1997), which is hereby incorporated by reference in itsentirety). Naturally occurring somatostatin and its analogs exhibitdifferential binding to these receptor subtypes, allowing precisetargeting of a peptide analog to specific diseased tissues.

In accordance with this aspect of the invention, the first and secondtargeting components have at least one biological activity of nativesomatostatin; preferably, this activity is the ability to specificallybind to a somatostatin receptor on a somatostatin receptor-bearing cell.Many such analogs having biological activity are known and have beendescribed, for example, in U.S. Pat. No. 5,770,687 to Hornik et al.;U.S. Pat. No. 5,708,135 to Coy et al.; U.S. Pat. No. 5,750,499 to Hoegeret al; U.S. Pat. No. 5,620,675 to McBride et al.; U.S. Pat. No.5,633,263 to Coy et al; U.S. Pat. No. 5,597,894 to Coy et al; U.S. Pat.No. 5,073,541 to Taylor et al; U.S. Pat. No. 4,904,642 to Coy et al;U.S. Pat. No. 6,017,509 to Dean; WO 98/47524 to Hoffman et al.; and U.S.Pat. No. 5,411,943 to Bogden, each of which is hereby incorporated byreference in its entirety.

In one embodiment, the first and second targeting components target thesomatostatin receptor-2.

In another embodiment of the present invention the cancer is breastcancer.

In accordance with this embodiment of the present invention, when thecancer is breast cancer, the first and second targeting componentstarget the HER receptor family.

First and second agents, as well as targeting and therapeuticcomponents, are described above.

In another embodiment of the present invention the cancer isnon-Hodgkin's Lymphoma.

In accordance with this embodiment, when the cancer is non-Hodgkin'slymphoma, the first and second targeting components target CD20.

First and second agents, as well as targeting and therapeuticcomponents, are described above.

Another aspect of the present invention relates to a combinationtherapeutic for treating cancer. The combination therapeutic includes afirst agent comprising a first targeting component coupled to a firstcancer therapeutic component and a second agent comprising a secondtargeting component coupled to a second cancer therapeutic component.The first and second targeting components have differentbiodistributions and/or pharmacokinetics.

First and second agents, as well as targeting and therapeuticcomponents, are described above.

Pharmaceutical compositions containing agents for use in the methods ofthe present invention can include a pharmaceutically acceptable carrieras described infra, one or more active agents, and a suitable deliveryvehicle. Suitable delivery vehicles include, but are not limited to,viruses, bacteria, biodegradable microspheres, microparticles,nanoparticles, liposomes, collagen minipellets, and cochleates.

In one embodiment of the present invention, the pharmaceuticalcomposition or formulation containing an inhibitory nucleic acidmolecule (e.g., siRNA molecule) is encapsulated in a lipid formulationto form a nucleic acid-lipid particle as described in Semple et al.,“Rational Design of Cationic Lipids for siRNA Delivery,” Nature Biotech.28:172-176 (2010), WO2011/034798 to Bumcrot et al., WO2009/111658 toBumcrot et al., and WO2010/105209 to Bumcrot et al., which are herebyincorporated by reference in their entirety.

In another embodiment of the present invention, the delivery vehicle isa nanoparticle. A variety of nanoparticle delivery vehicles are known inthe art and are suitable for delivery of an inhibitor of the invention(see e.g., van Vlerken et al., “Multi-functional Polymeric Nanoparticlesfor Tumour-Targeted Drug Delivery,” Expert Opin. Drug Deliv.3(2):205-216 (2006), which is hereby incorporated by reference in itsentirety). Suitable nanoparticles include, without limitation,poly(beta-amino esters) (Sawicki et al., “Nanoparticle Delivery ofSuicide DNA for Epithelial Ovarian Cancer Cell Therapy,” Adv. Exp. Med.Biol. 622:209-219 (2008), which is hereby incorporated by reference inits entirety), polyethylenimine-alt-poly(ethylene glycol) copolymers(Park et al., “Degradable Polyethylenimine-alt-Poly(ethylene glycol)Copolymers As Novel Gene Carriers,” J Control Release 105(3):367-80(2005) and Park et al., “Intratumoral Administration of Anti-KITENINshRNA-Loaded PEI-alt-PEG Nanoparticles Suppressed Colon CarcinomaEstablished Subcutaneously in Mice,” J Nanosci. Nanotechnology (2010),which are hereby incorporated by reference in their entirety), andliposome-entrapped siRNA nanoparticles (Kenny et al., “NovelMultifunctional Nanoparticle Mediates siRNA Tumor Delivery,Visualization and Therapeutic Tumor Reduction In Vivo,” J. ControlRelease 149(2): 111-116 (2011), which is hereby incorporated byreference in its entirety). Other nanoparticle delivery vehiclessuitable for use in the present invention include microcapsule nanotubedevices disclosed in U.S. Patent Publication No. 2010/0215724 to Prakashet al., which is hereby incorporated by reference in its entirety.

In another embodiment of the present invention, the pharmaceuticalcomposition is contained in a liposome delivery vehicle. The term“liposome” means a vesicle composed of amphiphilic lipids arranged in aspherical bilayer or bilayers. Liposomes are unilamellar ormultilamellar vesicles which have a membrane formed from a lipophilicmaterial and an aqueous interior. The aqueous portion contains thecomposition to be delivered. Cationic liposomes possess the advantage ofbeing able to fuse to the cell wall. Non-cationic liposomes, althoughnot able to fuse as efficiently with the cell wall, are taken up bymacrophages in vivo.

Several advantages of liposomes include: their biocompatibility andbiodegradability, incorporation of a wide range of water and lipidsoluble drugs; and they afford protection to encapsulated drugs frommetabolism and degradation. Important considerations in the preparationof liposome formulations are the lipid surface charge, vesicle size, andthe aqueous volume of the liposomes.

Liposomes are useful for the transfer and delivery of active ingredientsto the site of action. Because the liposomal membrane is structurallysimilar to biological membranes, when liposomes are applied to a tissue,the liposomes start to merge with the cellular membranes and as themerging of the liposome and cell progresses, the liposomal contents areemptied into the cell where the active agent may act.

Methods for preparing liposomes for use in the present invention includethose disclosed in Bangham et al., “Diffusion of Univalent Ions Acrossthe Lamellae of Swollen Phospholipids,” J. Mol. Biol. 13:238-52 (1965);U.S. Pat. No. 5,653,996 to Hsu; U.S. Pat. No. 5,643,599 to Lee et al.;U.S. Pat. No. 5,885,613 to Holland et al.; U.S. Pat. No. 5,631,237 toDzau & Kaneda, and U.S. Pat. No. 5,059,421 to Loughrey et al., which arehereby incorporated by reference in their entirety.

In another embodiment of the present invention, the delivery vehicle isa viral vector. Viral vectors are particularly suitable for the deliveryof inhibitory nucleic acid molecules, such as siRNA or shRNA molecules,but can also be used to deliver molecules encoding an anti-integrinantibody. Suitable gene therapy vectors include, without limitation,adenoviral vectors, adeno-associated viral vectors, retroviral vectors,lentiviral vectors, and herpes viral vectors.

Adenoviral viral vector delivery vehicles can be readily prepared andutilized as described in Berkner, “Development of Adenovirus Vectors forthe Expression of Heterologous Genes,” Biotechniques 6:616-627 (1988),Rosenfeld et al., “Adenovirus-Mediated Transfer of a Recombinant Alpha1-Antitrypsin Gene to the Lung Epithelium In Vivo,” Science 252:431-434(1991), WO 93/07283 to Curiel et al., WO 93/06223 to Perricaudet et al.,and WO 93/07282 to Curiel et al., which are hereby incorporated byreference in their entirety. Adeno-associated viral delivery vehiclescan be constructed and used to deliver an inhibitory nucleic acidmolecule of the present invention to cells as described in Shi et al.,“Therapeutic Expression of an Anti-Death Receptor-5 Single-Chain FixedVariable Region Prevents Tumor Growth in Mice,” Cancer Res. 66:11946-53(2006); Fukuchi et al., “Anti-Aβ Single-Chain Antibody Delivery viaAdeno-Associated Virus for Treatment of Alzheimer's Disease,” Neurobiol.Dis. 23:502-511 (2006); Chatterjee et al., “Dual-Target Inhibition ofHIV-1 In Vitro by Means of an Adeno-Associated Virus Antisense Vector,”Science 258:1485-1488 (1992); Ponnazhagan et al., “Suppression of HumanAlpha-Globin Gene Expression Mediated by the RecombinantAdeno-Associated Virus 2-Based Antisense Vectors,” J. Exp. Med.179:733-738 (1994); and Zhou et al., “Adeno-associated Virus 2-MediatedTransduction and Erythroid Cell-Specific Expression of a HumanBeta-Globin Gene,” Gene Ther. 3:223-229 (1996), which are herebyincorporated by reference in their entirety. In vivo use of thesevehicles is described in Flotte et al., “Stable in Vivo Expression ofthe Cystic Fibrosis Transmembrane Conductance Regulator With anAdeno-Associated Virus Vector,” Proc. Nat'l. Acad. Sci. 90:10613-10617(1993) and Kaplitt et al., “Long-Term Gene Expression and PhenotypicCorrection Using Adeno-Associated Virus Vectors in the Mammalian Brain,”Nature Genet. 8:148-153 (1994), which are hereby incorporated byreference in their entirety. Additional types of adenovirus vectors aredescribed in U.S. Pat. No. 6,057,155 to Wickham et al.; U.S. Pat. No.6,033,908 to Bout et al.; U.S. Pat. No. 6,001,557 to Wilson et al.; U.S.Pat. No. 5,994,132 to Chamberlain et al.; U.S. Pat. No. 5,981,225 toKochanek et al.; U.S. Pat. No. 5,885,808 to Spooner et al.; and U.S.Pat. No. 5,871,727 to Curiel, which are hereby incorporated by referencein their entirety.

Retroviral vectors which have been modified to form infectivetransformation systems can also be used to deliver a nucleic acidmolecule to a target cell. One such type of retroviral vector isdisclosed in U.S. Pat. No. 5,849,586 to Kriegler et al., which is herebyincorporated by reference. Other nucleic acid delivery vehicles suitablefor use in the present invention include those disclosed in U.S. PatentPublication No. 20070219118 to Lu et al., which is hereby incorporatedby reference in its entirety.

Regardless of the type of infective transformation system employed, itshould be targeted for delivery of the nucleic acid to the desired celltype. For example, for delivery into a cluster of cells (e.g., cancercells) a high titer of the infective transformation system can beinjected directly within the site of those cells so as to enhance thelikelihood of cell infection. The infected cells will then express theinhibitory nucleic acid molecule targeting the inhibition of integrinexpression. The expression system can further contain a promoter tocontrol or regulate the strength and specificity of expression of thenucleic acid molecule in the target tissue or cell.

Effective doses of the compositions of the present invention, for thetreatment of a metastatic disease vary depending upon many differentfactors, including type and stage of cancer, means of administration,target site, physiological state of the patient, other medications ortherapies administered, and physical state of the patient relative toother medical complications. Treatment dosages need to be titrated tooptimize safety and efficacy.

EXAMPLES

The examples below are intended to exemplify the practice of embodimentsof the disclosure but are by no means intended to limit the scopethereof

Materials and Methods

Internalization assay. LNCaP and CWR22Rv1 cells were trypsinized andaliquoted into tubes at 1×10⁶ cells per tube. Cells were washed oncewith RPMI (no FBS). ¹⁷⁷Lu-J591 (150,000 CPM) in 2004 RPMI (no FBS) pertube and/or ¹⁷⁷Lu-PSMA-617 (110,000 CPM) was added in 2004 RPMI (no FBS)per tube. The samples were incubated for 1 hour at 37° C., with thecells re-suspended every 15 minutes. After incubation, the cells werewashed twice with 0.1% BSA/PBS to remove unbound/un-internalized bindingagents. Samples were counted.

LNCaP xenografts. Each BALB/c^(nu/nu) male mouse was implantedsubcutaneously with 5 million LNCaP cells (with Matrigel). Ten dayslater, mice were randomly sorted into groups of 5 with similar tumorvolume. On day 0, 177 Lu-J591, 177 Lu-PSMA617, or PBS was injected viatail vein at various doses. Tumor volume and mouse weight were measured2-3 times per week. Tumor volume was calculated using the formula0.52*L*L*W and plotted against time (days post-treatment).

CWR22Rv1 xenografts. Each BALB/c^(nu/nu) male mouse was implantedsubcutaneously with 5 million CWR22Rv1 cells (with Matrigel). Four dayslater, mice were sorted into groups of 5 to achieve similar tumorvolume. ¹⁷⁷Lu-J591, 177 Lu-PSMA617, or PBS was injected via tail vein atthe indicated doses. Tumor volume and mouse weight were measured 2-3times per week. Tumor volume was calculated using the formula 0.52*L*L*Wand plotted against time (days post-treatment).

Example 1—Co-Incubation of Radiolabeled Anti-PSMA Antibody J591-¹⁷⁷Luand Radiolabeled PSMA-617-¹⁷⁷Lu Results in Additive ¹⁷⁷LuInternalization in PSMA-Positive Cells In Vitro

The individual agents, J591-¹⁷⁷Lu and PSMA-617-¹⁷⁷Lu, each internalizedto a similar degree within either given cell line (LNCaP and CWR22Rv1)(FIGS. 1A-1B). Internalization was greater in LNCaP than CWR22Rv1,consistent with the higher PSMA expression in the LNCaP cell line. Whenboth agents were co-incubated, both co-internalized effectively leadingto an additive amount of radiolabel within the cells.

Example 2—Combined Treatment Using Radiolabeled Anti-PSMA AntibodyJ591-¹⁷⁷Lu and Radiolabeled PSMA-617-¹⁷⁷Lu Results in AdditiveAnti-Tumor Effect In Vivo

In the LNCaP xenografts, PBS-treated animals (placebo control) developedtumors of approximately 1 cm in diameter by day 25 and requiredsacrifice several days later. The best anti-tumor effect was achieved byJ591-¹⁷⁷Lu at 150 μCi (FIG. 2 ). Equal anti-tumor effect could beachieved at half the dose of J591-¹⁷⁷Lu (i.e., 75 μCO by also adding adose of PSMA-617-¹⁷⁷Lu (i.e., 200 μCi) well below its MTD (FIG. 2 ).

In the CWR22Rv1 xenografts, placebo (PBS)-treated mice requiredsacrifice by day 17. J591-¹⁷⁷Lu, at the MTD dose of 180 μCi,demonstrated the best anti-tumor response (FIG. 3 ). This response couldbe equaled by J591-¹⁷⁷Lu at 135 μCi (75% of MTD) plus 525 μCi (a sub-MTDdose) of PSMA-617-¹⁷⁷Lu (FIG. 3 ).

Discussion of Examples 1-2

Taken together, these data show that co-administering 2 agents that binddifferent sites of a target molecule (e.g., a cell surface receptormolecule) present on a population of cells results in additive bindingof those agents. Where the target molecule/receptor is internalized,additive amounts of the 2 agents will, therefore, be internalized.Furthermore, if the 2 targeting agents have different properties (e.g.,molecular mass, charge, hydrophilicity/hydrophobicity, pharmacokinetics,bio-distribution, etc.) such that their respective side effects differ,and are non-overlapping, the 2 agents can be co-administered to resultin additive binding/uptake by the targeted cells without causing anyadded toxicity. As a corollary, the dose of each agent can be modestlyreduced (by 5-50%) below its respective maximum tolerated dose wherebythe co-administration of the 2 targeting agents still provides anadditive dose to the target cells but can substantially or completelyreduce the toxicity experienced by the subject.

Prophetic Example 1—Dosing in Human Patients

Tumor-related molecular targets, exemplified by prostate-specificmembrane antigen (PSMA), may be targeted in vitro and in vivo either byantibodies or small molecular ligands. These 2 classes of agents differgreatly both in molecular size and plasma half-life.

Antibody Ligand Size (molecular weight) 150,000 1,400* Plasma half-life3-7 days 3-7 hours *includes linker and chelate

As a result of the difference in size and its impact on vascularpermeability and ability to penetrate normal tissues, thebio-distribution of these two different classes or types of tumortargeting agents differs. The small molecular weight ligands rapidlyextravasate out of the blood vessels, into the extravascular fluid spaceand into the tissues with little to no barrier to entry. Conversely,large molecular weight molecules such as antibodies (Abs) circulate inthe vascular compartment for days to weeks. These far larger agents cantraverse reticuloendothelial organs such as the bone marrow and lymphnodes easily but other normal tissue barriers are traversed only slowlyparticularly in those normal tissues where there are basement membranes,intervening cell layers and epithelial tight junctions. In the case ofinvasive tumors, these normal barriers are breached by the tumor cellsthemselves making the tumor cells readily accessible to Abs.

Size also plays a role in how long a molecule stays in the body beforeit is excreted. For instance, small molecules very easily pass throughthe kidney glomerulus and are promptly excreted in the urine. This isthe case with the small molecule ligands such as those that target PSMAand somatostatin type 2 receptors (SSTR-2). Abs, however, are far toolarge to pass through the glomerulus so they are not excreted by theurinary tract, stay much longer in the body and are more oftenmetabolized by the liver.

While the two different classes of agents both bind to their target(e.g., PSMA, SSTR-2, CD20, etc)-expressing tumor cells, the normaltissues that get targeted differs between classes of agents. As anexample, PSMA is expressed not only by prostate cancer cells but also bythe normal parotid and other salivary glands, lacrimal glands, kidneyand small bowel (See FIGS. 4-7 ). Small molecule/inhibitor-based PSMAagents target these normal tissue sites—parotid and other salivaryglands, lacrimal glands, kidneys and small bowel; anti-PSMA Abs do nottarget the salivary or other glands nor the kidneys as the Abs are toolarge to penetrate into these normal tissues. The differentbio-distributions of these 2 types of agents, large Abs and smallligands, is demonstrated by imaging scans of patients who wereadministered each type of agent (FIG. 8 and FIG. 9 ). With both types ofagents, the tumor sites are targeted, but the normal tissues that aretargeted are different and virtually mutually exclusive.

By targeting tumors with both of the 2 different types of agents, thetumor gets additive dosing while the non-target normal tissues do notreceive additive doses. Another advantage of targeting with 2 differenttypes of agents is that one has the option to use more than 1 type ofcytotoxin. For example, with radioisotope therapeutics, one can targetan alpha and a beta or 2 alphas or 2 beta particles.

Prophetic Example 2—Beta+Beta

In the case of targeting a beta (e.g., ¹⁷⁷Lu) on an Ab, e.g., J591-Lu¹⁷⁷to target PSMA, the key (and dose-limiting) side effect isthrombocytopenia (decreased platelet count), and the relative degree ofthrombocytopenia progressively increases in magnitude as the doseincreases. While none of the patients treated with J591-Lu¹⁷⁷ sufferedany bleeding episodes, some did require platelet transfusions untiltheir platelet counts naturally recovered. No patients at doses of 30mCi/m²×2 doses (approx. 120 mCi total in a man of approx. 2.0 m 2) or 35mCi/m²×2 doses (approx. 140 mCi total) required platelet transfusions,whereas 5 of 16 (31%), and 9 of (60%) patients at 40 mCi/m²×2 doses(approx. 160 mCi total) and 45 mCi/m²×2 doses (approx. 180 mCi total)doses, respectively, did require platelet transfusions to support themwhile their bone marrow recovered.

Total dose/activity of # of patients platelet J591-Lu administeredtransfusions(%) 120 0 140 0 160 5/16 (31%) 180 9/16 (60%)

J591-Lu¹⁷⁷ begins to induce PSA declines, as an indication of anti-tumoractivity, at an administered ¹⁷⁷Lu dose of ≤30 mCi/m² given twice at a 2week interval. As the dose is increased to 140, 160 and 180 mCi [total],the magnitude of the PSA/anti-tumor response gets progressively greater.

Kabasakal et. al., “Pre-Therapeutic Dosimetry of Normal Organs andTissues of ¹⁷⁷Lu-PSMA-617 Prostate-Specific Membrane Antigen (PSMA)Inhibitor in Patients With Castration-Resistant Prostate Cancer,” Eur.J. Nucl. Med. Mol. Imaging 42:1976-1983 (2015), which is herebyincorporated by reference in its entirety, and Kratochwil et. al.,“PSMA-Targeted Radionuclide Therapy of Metastatic Castration-ResistantProstate Cancer With ¹⁷⁷Lu-Labeled PSMA-617,” J. Nucl. Med. 57:1170-1176(2016), which is hereby incorporated by reference in its entirety, havedetermined that the maximum safe cumulative dose of PSMA-617-Lu¹⁷⁷ (aligand representative of PSMA-binding small moleculeligands/inhibitors/peptides) is 27-30 Gbq as the limit to the kidney andsalivary glands. Doses at this level or beyond subject the patients topotential kidney function compromise and xerostomia (dry mouth). Severexerostomia results in loss of taste and appetite and severe dental/oralcavity disease. Patients complain about difficulty chewing, swallowing,sleeping, speaking, abnormal taste and a burning sensation in the mouth.

PSMA-617/ligand-Lu¹⁷⁷ begins to induce PSA declines at an administeredcumulative ¹⁷⁷Lu dose of 300 mCi (11.1 Gbq) given in a 2-dose regimen ata 2 week interval (150 mCi×2). As the dose is increased to 2-dose totalsof 400-500 mCi (14.8-18.5 GBq), the magnitude of the PSA/anti-tumorresponse gets progressively greater.

J591-¹⁷⁷Lu does not deliver large radioactive doses to the kidneys or tothe salivary glands, and PSMA-617/ligand does not deliver large doses tothe bone marrow so it does not cause thrombocytopenia—that is, these twodifferent targeting agents both lead to anti-tumor activity but theirrespective side effects are mutually exclusive.

The proposed therapeutic approach seeks to attain maximal dose to thetumor and resulting anti-tumor effect by targeting ¹⁷⁷Lu (or othercytotoxic agents known to those in the art) using a combination of the 2respective agents: anti-PSMA Ab (e.g., J591) and PSMAligand/inhibitor/peptide (e.g., PSMA-617, PSMA-11, PSMA I & T, RM-2,etc). By modestly lowering the dose of each agent, the side effects toplatelets [due to anti-PSMA Ab] and to kidneys and salivary glands [dueto PSMA ligand/inhibitor/peptide] can be reduced or avoided. Morespecifically, a dose of J591-¹⁷⁷Lu 120-140 mCi total in a 2 week cycleis proposed. Another possible dose range of J591-¹⁷⁷Lu is from 100-160mCi total in a 2 week cycle. The J591-¹⁷⁷Lu dosing is to be accompaniedby contemporaneous dosing of PSMA ligand/inhibitor/peptide (e.g.,PSMA-617) of 400 mCi total in a 2 week cycle (or a range of 300-500 mCitotal in a 2 week cycle). At these doses, no significant salivary glandor kidney function compromise occurs, and the doses are below the 27-30Gbq cumulative doses determined by Kabasakal et. al., “Pre-TherapeuticDosimetry of Normal Organs and Tissues of ¹⁷⁷Lu-PSMA-617Prostate-Specific Membrane Antigen (PSMA) Inhibitor in Patients WithCastration-Resistant Prostate Cancer,” Eur. J. Nucl. Med. Mol. Imaging42:1976-1983 (2015), which is hereby incorporated by reference in itsentirety, and Kratochwil et. al., “PSMA-Targeted Radionuclide Therapy ofMetastatic Castration-Resistant Prostate Cancer With ¹⁷⁷Lu-LabeledPSMA-617,” J. Nucl. Med. 57:1170-1176 (2016), which is herebyincorporated by reference in its entirety. If desired, 1-2 additionaldoses of 200-250 mCi (7.4-9.25 Gbq) could be given at ≥2 week interval/sand still remain below the 27-30 Gbq limit.

Prophetic Example 3—Alpha+Alpha

Alpha particles offer significantly more cell killing potential thanbetas due to their substantially greater atomic mass, linear energytransfer and radiobiological effect. The potential of substantialanti-tumor effect when treating prostate cancer patients withPSMA-617-Ac²²⁵ has been reported. Unfortunately, this treatment islimited by the intolerable damage done to the salivary glands. Treatmentactivities of 50 kBq/kg were without toxicity but induced insufficientanti-tumor response in patients with high tumor burden. However, anincrease in administered activity led to severe xerostomia becoming thedose-limiting toxicity if treatment activity exceeded 100 kBq/kg percycle (Kratochwil et. al., “Targeted Alpha-Therapy of MetastaticCastration-Resistant Prostate Cancer With (225)Ac-PSMA-617: DosimetryEstimate and Empiric Dose Finding,” J. Nucl. Med. 58(10):1624-1631(2017), which is hereby incorporated by reference in its entirety).Although greater anti-tumor activity occurred at higher doses, the sideeffects were too great. However, this can be improved by deliveringadditional Ac²²⁵ to the tumor via the Ab which does not target thesalivary glands. The appropriate dose of J591-Ac²²⁵ can be determined ina standard phase 1 trial in which all patients receive PSMA 617-Ac²²⁵ atthe tolerable dose of 100 Kbq/kg plus escalating doses of J591-Ac²²⁵until an MTD is determined. This will result in determination of thedosing of the combination of PSMA ligand-Ac²²⁵+anti-PSMA Ab-Ac²²⁵.

Prophetic Example 4—Alpha+Beta

Combining treatment with an alpha particle and its high energy and highcytotoxicity as well as its focused short range along with a beta, whichhas a longer range, also offers patient benefit as the latter willprovide anti-tumor activity at the tumor margins. It is preferred totarget the alpha using the Ab so as to avoid the intolerable salivarygland side effects and target the beta using the PSMA ligand to avoidserious hematological toxicity. All patients would receive PSMA617-Lu¹⁷⁷ at 400 mCi total in a 2 week cycle (or a range of 300-500 mCitotal in a 2 week cycle). The appropriate dose of J591-Ac²²⁵ can bedetermined in a standard phase 1 trial format in which escalating dosesof J591-Ac²²⁵ are combined with the stated dose of PSMA-617-¹⁷⁷Lu aboveuntil an MTD is determined.

Alternatively, the ¹⁷⁷Lu can be targeted by the Ab and Ac²²⁵ byPSMA-617. In this case, the latter is dosed at 100 kbq/kg, and the theAb J591 is dosed at J591-¹⁷⁷Lu 120-140 mCi total in a 2 week cycle.Another possible dose range of J591-¹⁷⁷Lu is from 100-160 mCi total in a2 week cycle.

Although preferred embodiments have been depicted and described indetail herein, it will be apparent to those skilled in the relevant artthat various modifications, additions, substitutions, and the like canbe made without departing from the spirit of the invention and these aretherefore considered to be within the scope of the invention as definedin the claims which follow.

1. (canceled)
 2. A method for treating prostate cancer in a subject, themethod comprising co-administering to a subject in need thereof, atherapeutically effective amount of J591-Ac²²⁵ and a therapeuticallyeffective amount of a PSMA ligand/inhibitor.
 3. The method of claim 2,wherein the PSMA ligand/inhibitor is a PSMA-Lu¹⁷⁷ peptide.
 4. The methodof claim 3, wherein the PSMA-Lu¹⁷⁷ peptide is PSMA I&T-Lu¹⁷⁷ or PSMA617-Lu¹⁷⁷.
 5. The method of claim 3, wherein the J591-Ac²²⁵ andPSMA-Lu¹⁷⁷ peptide are administered in separate compositions.
 6. Themethod of claim 5, wherein the separate compositions are administered tothe subject by injection.
 7. The method of claim 5, wherein the separatecompositions are co-administered via simultaneous administration,sequential administration, continuous administration, or a combinationthereof to the subject.
 8. The method of claim 7, wherein the separatecompositions are administered in a 2 week cycle.
 9. The method of claim2, wherein J591-Ac²²⁵ and PSMA ligand/inhibitor are co-administered tothe subject to allow for J591-Ac²²⁵ and PSMA ligand/inhibitor to bedosed at about 25% to about 100% of their respective maximum tolerateddoses.
 10. The method of claim 9, wherein the PSMA ligand/inhibitor is aPSMA-Lu¹⁷⁷ peptide.
 11. The method of claim 10, wherein the PSMA-Lu¹⁷⁷peptide is PSMA I&T-Lu¹⁷⁷ or PSMA 617-Lu¹⁷⁷.
 12. A combinationtherapeutic comprising a therapeutically effective amount of J591-Ac²²⁵and a therapeutically effective amount of a PSMA ligand/inhibitor. 13.The combination therapeutic of claim 12, wherein the PSMAligand/inhibitor is a PSMA-Lu¹⁷⁷ peptide.
 14. The combinationtherapeutic of claim 13, wherein the PSMA-Lu¹⁷⁷ peptide is PSMA617-Lu¹⁷⁷ or PSMA I&T-Lu¹⁷⁷.
 15. The combination therapeutic of claim12, wherein the J591-Ac²²⁵ and the PSMA ligand/inhibitor are in the sameor in separate compositions.
 16. The combination therapeutic of claim15, wherein the J591-Ac²²⁵ and the PSMA ligand/inhibitor are in separatecompositions.
 17. The combination therapeutic of claim 16, wherein theseparate compositions are formulated for injection to a subject.
 18. Thecombination therapeutic of claim 16, wherein the separate compositionsare formulated for co-administration via simultaneous administration,sequential administration, continuous administration, or a combinationthereof to a subject.
 19. The combination therapeutic of claim 16,wherein the separate compositions are administered to a subject in a 2week cycle.
 20. The combination therapeutic of claim 12, wherein thecombination therapeutic is formulated for administration to a subject todeliver the J591-Ac²²⁵ and PSMA ligand/inhibitor peptide at a dose ofabout 25% to about 100% of their respective maximum tolerated doses. 21.The combination therapeutic of claim 20, wherein the PSMAligand/inhibitor is a PSMA-Lu¹⁷⁷ peptide.
 22. The combinationtherapeutic of claim 21, wherein the PSMA-Lu¹⁷⁷ peptide is PSMA617-Lu¹⁷⁷ or PSMA I&T-Lu¹⁷⁷.